The present invention relates to compositions and methods involving IKKxcex3 and IKKxcex3 mutants. In particular, the present invention provides methods and compositions, including transgenic animals, suitable for use in determining means to treat, control, and/or prevent incontinentia pigmenti (IP). The present invention also provides methods to detect the presence of mutations in the IKKxcex3 gene and protein.
Incontinentia pigmenti (IP), an X-linked human genodermatosis is a relatively rare disorder associated with multiple congenital defects (Landy and Donnai, J. Med. Genet. 30:53-59 [1993]; and Francis and Sybert, Semin. Cutan. Med. Surg. 16:54-60 [1997]), the gene of which (IP) has been mapped to Xq28 (Sefiani et al., Genomics 4:427-429 [1989]; and Sefiani et al., Human. Genet. 86:297-299 [1991]). Because of Lyonization, IP occurs almost exclusively in females, as most affected males die pre- and perinatally, unless their karyotype is 47XXY (Landy and Donnai, supra [1993]; Francis and Sybert, supra [1997]; and Scheuerle, Am. J. Med. Genet. 77:201-218 [1998]). The characteristic features of IP are detected at or soon after birth. These features commonly begin with an erythematous eruption of the skin, with linear vesiculation. The blistering stage progresses to the verrucous stage, in which multiple longitudinal verrucous lesions are distributed along the skin. Within a year, these hyperkeratotic lesions disappear and leave behind the classic hyperpigmented whorls and streaks, which may fade later in life. The name of the disease is derived from the characteristic finding that these dark lines and swirls are due to loss (incontinence) of melanin from basal keratinocytes and its deposition as free pigment or within dermal macrophages (i.e., melanophages). The first three stages of the disease occasionally overlap. Eventually, many of the cutaneous symptoms disappear and the disease in adult females (i.e., subjects who can pass the disease along to their progeny) is characterized by irregular, pale, hairless, anhidrotic streaks and splashes of hyperpigmentation, resulting in a xe2x80x9cmarble cakexe2x80x9d pattern (Wettke-Scahfer and Kantner, Hum. Genet. 64:1-23 [1983]; Landy and Donnai, supra [1993]; and Francis and Sybert, supra [1997]).
Although this disease is rare, the significant morbidity and mortality associated with IP indicate the need to develop methods to treat and prevent the disease.
The present invention relates to compositions and methods involving both wild type and mutant IxcexaB kinase-xcex3 (IKKxcex3) genes and proteins. In particular, the present invention provides methods and compositions, including transgenic animals, suitable for use in determining means to treat, control, and/or prevent the genodermatosis, incontinentia pigmenti (IP).
IKKxcex3, also known as the nuclear factor-xcexaB (NF-xcexaB) essential modulator or NEMO, is the essential regulatory subunit of the IxcexaB kinase (IKK) and is encoded by an X-linked gene in mice and humans. It is required for NF-xcexaB activation and resistance to tumor necrosis factor (TNF)-induced apoptosis. Female mice heterozygous for IKKxcex3/NEMO deficiency develop unique dermatopathy, characterized by keratinocyte hyperproliferation, skin inflammation, hyperkeratosis, and increased apoptosis. Although Ikkxcex3+/xe2x88x92 females eventually recover, Ikkxcex3xe2x88x92 males die in utero. These symptoms and inheritance pattern are very similar to those of IP, a human genodermatosis that is syntenic with the IKKxcex3/NEMO locus. Indeed, biopsies and cells from IP patients exhibit defective IKKxcex3/NEMO expression, but normal expression of IKK catalytic subunits. This unique self-limiting disease, the first to be genetically linked to the IKK signaling pathway, is dependent upon X-chromosome inactivation. Although an understanding of the mechanism(s) is not necessary in order to use the present invention, the results obtained during the development of the present invention indicate that IKKxcex3/NEMO-deficient cells trigger an inflammatory reaction that eventually leads to the death of these cells.
The present invention provides transgenic nonhuman animals having a genome comprising a disruption of the endogenous Ikkxcex3/NEMO gene, which is a result of the insertion of a transgene and which causes a decrease in IKKxcex3/NEMO expression. In some embodiments, IKKxcex3/NEMO expression is eliminated. In a preferred embodiment, the transgenic nonhuman animal is a mouse bearing a heterozygous disruption of the Ikkxcex3/NEMO gene. In some embodiments, the transgenic mouse exhibits hypersensitivity to TNFxcex1-induced apoptosis, while in other embodiments, the transgenic mouse exhibits dermatopathy, keratinocyte hyperproliferation, skin inflammation and/or hyperkeratosis.
The present invention further provides cells derived from a transgenic nonhuman animal having a genome comprising a disruption of the endogenous Ikkxcex3/NEMO gene, wherein the disruption is a result of the insertion of a transgene. In particularly preferred embodiments, the disruption results in a decrease in IKKxcex3/NEMO expression. In one preferred embodiment, the cell is derived from a transgenic mouse bearing a heterozygous disruption of the Ikkxcex3/NEMO gene. In some embodiments, the cell is an embryonic stem cell, while in other embodiments, the cell is selected from the group consisting of embryonic fibroblasts, hepatocytes, thymocytes, splenocytes and epidermal cells. In some preferred embodiments, the cell exhibits hypersensitivity to TNFxcex1-induced apoptosis.
The present invention also provides methods for screening for biologically active agents to treat incontinentia pigmenti. These methods comprise: (a) exposing a transgenic mouse having a genome comprising a disruption of the endogenous Ikkxcex3/NEMO gene, which is a result of the insertion of a transgene and which causes a decrease in IKKxcex3/NEMO expression to a candidate agent; and (b) determining the effect of the candidate agent on incontinentia pigmenti pathology. In some embodiments, assessment of the effect of the candidate agent on incontinentia pigmenti pathology is accomplished by measuring sensitivity to TNFxcex1-induced apoptosis, while in other embodiments the degree of dermatopathy, keratinocyte hyperproliferation and/or skin inflammation is assessed.
The present invention also provides methods for detecting mutant Ikkxcex3/NEMO genes in biopsy material obtained from individuals. These methods comprise detecting IKKxcex1 and IKKxcex2 expression, in the absence of IKKxcex3/NEMO expression. In some embodiments, detection is by immunoblot, while in others it is by Northern blot, Southern blot, reverse transcription-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis and/or conformation-sensitive gel electrophoresis (CSGE).